US20100099131A1 - Solution of matrix - Google Patents

Solution of matrix Download PDF

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Publication number
US20100099131A1
US20100099131A1 US12/449,723 US44972308A US2010099131A1 US 20100099131 A1 US20100099131 A1 US 20100099131A1 US 44972308 A US44972308 A US 44972308A US 2010099131 A1 US2010099131 A1 US 2010099131A1
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Prior art keywords
acetonitrile
water
matrix
solution
concentration
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Abandoned
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US12/449,723
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English (en)
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Wataru Hattori
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NEC Corp
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NEC Corp
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Publication of US20100099131A1 publication Critical patent/US20100099131A1/en
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates

Definitions

  • the present invention relates to a solution of a matrix for being discharged through a small size nozzle.
  • the mass spectrometric imaging is a technology for allowing laser beam to impinge on tissue slices or specimens isolated with chips or gel at the respective positions to conduct a mass spectrometry for each of the impinging locations.
  • Such technology requires the procedures, in which a reagent for accelerating an ionization of the specimen such as protein and the like, which is referred to as “matrix”, is added to the specimen before the impingement with laser beam to present a condition of a crystallization of the specimens with the matrix.
  • matrix reagent for accelerating an ionization of the specimen
  • higher positional resolution is more preferable in such technology, though the positional resolution is limited by the following factors.
  • the first factor is a diameter of a spot of an impinging laser beam.
  • the diameter of the laser beam spot in general MALDI-MS is around 100 microns to 200 microns.
  • the second factor is an unevenness of a specimen distribution in case of adding the matrix.
  • the matrix is ordinarily dissolved in a mixed solvent of a volatile organic solvent (acetonitrile) and water to be added in tissue slices or a chip substrate in a form of a solution, and a specimen such as a protein presented there is taken when the matrix is precipitated or crystallized due to a dehydration of a solvent.
  • acetonitrile acetonitrile
  • water acetonitrile
  • a specimen such as a protein presented there is taken when the matrix is precipitated or crystallized due to a dehydration of a solvent.
  • an excessive amount of solution for the matrix causes that the matrix solution containing the specimen such as protein and the like is extensively spread over the tissue slice, over the chip or the gel, causing a disturbance on the positional information of specimen such as protein. Therefore, the technology for adding the matrix requires a technology for adding a smaller amount of a solution for every smaller area.
  • a small size nozzle such as, for example, an ink-jet, a dispenser, a spray, an electrospray, an ultrasonic atomizer device and the like is employed to add the matrix.
  • a diameter of a droplet or an inner diameter of a small size nozzle is substantially equivalent to, or smaller than, a spot diameter of laser beam for the MALDI-MS.
  • a quantity of a droplet of the matrix solution is in the level of nano-liter (nL).
  • Sinapinic acid is versatilely employed for the matrix, not only in the case of employing for the imaging technology, but also in the case of conducting an ordinary mass spectrometry.
  • Sinapinic acid is often utilized in a solution in the condition of super-saturation or at a concentration of 10 mg/mL, which is closer to the concentration of the saturated solution, according to a recommendation of manufacturers for mass spectrometers or reagents.
  • a solvent of a liquid mixture of acetonitrile 3: water 7 in a volumetric ratio is often employed, and such solvent often contains trifluoro acetic acid (TFA) at a concentration of 0.1 to 1%.
  • TFA trifluoro acetic acid
  • Molecular weight of sinapinic acid is 224.21, and thus an amount of a substance of sinapinic acid contained in 1 nL of a matrix solution of sinapinic acid at a concentration of 10 mg/mL is 44.6 pmol. Therefore, an amount of a specimen such as protein or peptide corresponding to the molar ratio of one-1,000th is 44.6 fmol. Given that the lower limit of the sensibility for the ordinary commercially available mass spectrometer is 10 fmol, such specimen concentration is almost the value of the lower limit for stable detection.
  • Patent Document 1 discloses results of mass spectrometric imaging by employing a matrix solution containing sinapinic acid dissolved at a concentration of 20 mg/mL in a solvent containing 1 acetonitrile and 1 water in volumetric ratio and also containing TFA at a concentration of 0.1 to 1%.
  • the conventional matrix solution is a saturated solution of sinapinic acid or a solution at a concentration extremely closer to the saturation. Therefore, when the matrix solution is discharged from a small size nozzle, sinapinic acid precipitates and crystallizes in the inside of the small size nozzle or at the discharging opening of the nozzle to cause a problem of plugging the small size nozzle.
  • a solution of a matrix for being discharged through a small size nozzle containing a liquid mixture consisting of acetonitrile and water as main solvent component, wherein a volumetric ratio of acetonitrile and water is within a range of from acetonitrile 6.5: water 3.5 to acetonitrile 8: water 2, and wherein sinapinic acid is contained as the matrix.
  • the matrix solution containing sinapinic acid dissolved therein is selected to be within a range of from acetonitrile 6.5: water 3.5 to acetonitrile 8: water 2, so that the matrix solution containing sinapinic acid at higher concentration can be obtained.
  • the matrix solution can be discharged through a small size nozzle without precipitating sinapinic acid therein. Therefore, an addition of the matrix solution in a form of smaller droplet can be conducted, thereby achieving a mass spectrometric imaging with enhanced positional resolution.
  • a matrix solution is provided, which is difficult to cause a plugging of a small size nozzle when the solution is discharged through the small size nozzle, while maintaining a sufficiently higher concentration of sinapinic acid frequently employed as a matrix in a sinapinic acid solution.
  • FIG. 1 is a graph, helpful in describing an advantageous effect of the present exemplary embodiment
  • FIG. 2 is a graph, helpful in describing an advantageous effect of the present exemplary embodiment.
  • FIG. 3 is a graph, showing results of the present example.
  • a matrix solution according to the present exemplary embodiment is a matrix solution to be discharged through a small size nozzle.
  • the solution contains a liquid mixture consisting of acetonitrile and water as main solvent component, and a volumetric ratio of acetonitrile and water is within a range of from acetonitrile 6.5: water 3.5 to acetonitrile 8: water 2, and sinapinic acid is contained as the matrix.
  • the principal solvent component of the matrix solution according to the present exemplary embodiment is a liquid mixture of acetonitrile and water at a volumetric ratio of acetonitrile and water of within a range of from acetonitrile 6.5: water 3.5 to acetonitrile 8: water 2.
  • the principal solvent component may be a constituent constituting equal to or higher than 90% of the solvent by volumetric ratio, and may more preferably be a constituent constituting equal to or higher than 97%.
  • the principal solvent component of the matrix solution according to the present exemplary embodiment may more desirably be a liquid mixture of acetonitrile and water at a volumetric ratio within a range of from acetonitrile 7: water 3 to acetonitrile 8: water 2.
  • the concentration of sinapinic acid is influenced by the sensibility of the mass spectrometry, and is not particularly limited provided that the concentration allows achieving the object of mass spectrometry, and preferably within a range of from 4.48 mg/mL to 11.2 mg/mL.
  • the matrix solution according to the present exemplary embodiment may contain a solvent, which is capable of being mixed with acetonitrile and water.
  • a polar solvent such as alcohol and the like, for example, may be employed for such solvent.
  • Trifluoro acetic acid for example, may be employed for such acid. While the concentration of acid is not particularly limited, the concentration may be, for example, within a range of from 0.1% to 1%, when TFA is employed.
  • the matrix solution according to the present exemplary embodiment is discharged through a small size nozzle.
  • the type of the small size nozzle is not particularly limited, typical example thereof may include an ink-jet, a dispenser, a spray, and an electrospray.
  • the matrix solution according to the present exemplary embodiment can be successfully discharged even if the solution is discharged through the small size nozzle having an internal diameter of the aperture of equal to or smaller than 0.18 mm. While the lower limit of the internal diameter of the aperture of the nozzle may not particularly limited provided that the aperture diameter is suitable for adding the matrix solution, the aperture diameter may be equal to or larger than 0.10 mm in view of a practicality, a commercial-availability and a cost.
  • the temperature in the discharging process is not particularly limited provided that the temperature is suitable for the operation of the small size nozzle, it is preferable for discharging at a temperature within a range of from 10 to 30 degrees C., and it is more preferable for discharging at a temperature within a range of from 20 to 25 degrees C.
  • FIG. 1 is a graph which measured saturated concentration of sinapinic acid for solvent with room temperature for solvents of different mixing ratios of acetonitrile and water.
  • Lower abscissa axis represents concentrations of acetonitrile
  • upper abscissa axis represents concentrations of water, both in volumetric percent (%).
  • Left ordinate axis represents concentrations of sinapinic acid in a unit of mg/mL
  • right ordinate axis represents the converted molar concentrations equivalent to the concentration represented by the right ordinate axis in a unit of pmol/mL.
  • the saturated concentration of sinapinic acid considerably varies with the mixing ratio of acetonitrile and water.
  • the saturated concentration is maximized in the case of the volumetric ratio of acetonitrile 7 and water 3 with a maximum value of 56.4 mg/mL.
  • the saturated concentrations in the cases of the volumetric ratio ranging from acetonitrile 6.5 and water 3.5 to acetonitrile 8 and water 2 are about 50 mg /mL or higher, which is equivalent to five folds of the saturated concentration of 10 mg/mL in the ordinary conditions.
  • sinapinic acid is difficult to precipitate by employing such condition of the volumetric ratio of acetonitrile and water when a solution of sinapinic acid is prepared at a concentration of 10 mg/mL, which is one-fifth of the saturated concentration, thereby preventing the needle from being plugged.
  • FIG. 2 is a graph, showing a temperature-vapor pressure curve of acetonitrile and water.
  • abscissa axis represents temperature (degree C.)
  • left ordinate axis represents vapor pressure in unit of mmHg
  • right ordinate axis represents the converted vapor pressure in unit of kPa.
  • acetonitrile exhibits a vapor pressure around 20 degrees C., which is about four folds of that of water. Therefore, the solvent evaporates in the gas-liquid interface at the tip of the small size nozzle according to such rate.
  • the volumetric ratio of the principal solvent of the matrix solution according to the present exemplary embodiment is closer to the above-described ratio of the vapor pressure. Therefore, it may be understood that such volumetric ratio presents a stable condition, which is not much changed by the drying.
  • the concentration of sinapinic acid in the solution may be equal to or higher than 4.48 mg/mL, namely equal to or higher than 20 pmol/ ⁇ L for achieving stably measurements, which corresponds to two folds of the sample amount for the lower limit in the sensibility for the commercially available mass spectrometer of 10 fmol.
  • the concentration may be equal to or lower than 11.2 mg/mL, which is not higher than fivefold of the saturated concentration. This allows adding sinapinic acid at a sufficient concentration for specimens serving as an object for the mass spectrometry and thereby preventing the needle from being plugged.
  • a metal needle for dispenser of 28G (internal diameter: 0.18 mm, external diameter: 0.36 mm) was employed to drop the respective matrix solutions prepared according to the above-described procedures at a room temperature to determine the discharging ability.
  • matrix solutions were also dropped again after a certain time was past to determine the re-discharging ability for the matrix solutions.
  • a protein of apomyoglobin was added to above-described prepared matrix solutions at a concentration of one-thousandth of the concentration of sinapinic acid in molar ratio to determine the solubility of apomyoglobin.
  • a metal needle for dispenser of 32G (internal diameter: 0.10 mm, external diameter: 0.23 mm) was employed to respectively drop the above-described prepared matrix solutions containing apomyoglobin at a room temperature along a flow path of a chip substrate having a straight flow path.
  • the substrate was then transferred to the mass spectrometer for measuring the signal detection intensity of apomyoglobin to evaluate the uniformity of the signal detection intensity.
  • the results are shown in Table 1.
  • the mark ⁇ represents goodness
  • the mark X represents failure
  • the mark ⁇ represents the conditions mixed up with goodness ( ⁇ ) and failure ( ⁇ ) resulting in unstable condition.
  • the condition of the volumetric ratio of acetonitrile and water of 6:4 provides clearly deteriorated uniformity, as compared with the other conditions of the volumetric ratios. Then, the experiment was continued by diluting the solution to have the concentration of sinapinic acid of about 8.5 mg/mL, and the result showed that improved uniformity for the signal detection intensity was obtained. Therefore, it was found that the crystals more easily precipitate in the needle in the condition of the volumetric ratio of acetonitrile and water of 6:4, as compared with the other conditions of the volumetric ratio, easily causing unstable discharging rate of the solution.
  • the saturated concentration of sinapinic acid is 43 mg/mL from the graph of FIG. 1 .
  • the concentration of sinapinic acid may be selected to be 8.5 mg/mL to obtain enhanced signal strength as described above, such that it was thought that the concentration of sinapinic acid should be not higher than 1/5 of the saturated concentration.
  • solutions of matrix of sinapinic acid were prepared with the solvent with volumetric ratio of acetonitrile and water of 7:3 and 8:2 while calculating the concentration of the matrix by mol concentration, and experiments for the apomyoglobin detection sensitivity with a mass spectrometer were conducted with the prepared solutions of the matrix, and it was found that clear signal could be detected when the concentration of sinapinic acid was not lower than 4.48 mg/mL (20 pmol/ ⁇ L).
  • the constitution of the matrix solution was optimized by such experiments to obtain the results, in which variations in the signal detection intensity for apomyoglobin were equal to or less than ⁇ 30% in the case of conducting the dropping operations along a flow path in a chip for the conditions of the volumetric ratio of /acetonitrile and water of 7:3 and 8:2. It was also confirmed that the detection was conducted within the flow path in the chip without disturbing a pattern of protein isolated by isoelectric focusing.
  • FIG. 3 is a graph, which includes mass spectrometry spectrums taken by every 0.5 mm in the flow path. Abscissa represents molecular weight, and ordinate represents signal strength. It is found that trypsin inhibitor, carbonic anhydrase II, and creatine phosphokinase, which are isolated in advance in the flow path in the chip by isoelectric focusing phoresis, were detected without particularly spreading through the inside of the flow path.
  • the matrix solution which hardly causes a plugging of the small diameter nozzle during the dropping process through the nozzle while the concentration of the solution of sinapinic acid frequently employed as the matrix is maintained at the necessary and sufficient concentration, can be provided.
  • the configuration is not limited to the dispenser, and it is needless to point out that the configuration of the example may also be generally adopted to the technology for adding the matrix solution with the small diameter nozzle.
US12/449,723 2007-02-28 2008-02-13 Solution of matrix Abandoned US20100099131A1 (en)

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JP2007-050429 2007-02-28
JP2007050429 2007-02-28
PCT/JP2008/000212 WO2008105142A1 (ja) 2007-02-28 2008-02-13 マトリックス溶液

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JP5365547B2 (ja) * 2010-02-25 2013-12-11 株式会社島津製作所 Ｍａｌｄｉ−ｍｓ用試料調製方法
KR20120090473A (ko) * 2011-02-08 2012-08-17 주식회사 아스타 말디 이미징용 매트릭스 용액 및 이를 이용한 생체 분자의 말디 이미징의 측정 방법

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US5580434A (en) * 1996-02-29 1996-12-03 Hewlett-Packard Company Interface apparatus for capillary electrophoresis to a matrix-assisted-laser-desorption-ionization mass spectrometer
US20030220447A1 (en) * 1996-09-26 2003-11-27 Shearwater Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
JP2005117251A (ja) * 2003-10-06 2005-04-28 Sanyo Electric Co Ltd 通信装置
US20070069122A1 (en) * 2005-06-03 2007-03-29 Angelique Augustin In situ polypeptide identification
US20070184524A1 (en) * 2000-11-20 2007-08-09 Gokarn Ravi R 3-hydroxypropionic acid and other organic compounds
US7452715B1 (en) * 1999-06-25 2008-11-18 Wyeth Holdings Corporation Production of the lipidated form of the peptidoglycan associated lipoproteins of gram-negative bacteria

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JP2006292680A (ja) * 2005-04-14 2006-10-26 Toyo Kohan Co Ltd 固体支持体上において相互作用した生体分子を分析する方法およびそのための固体支持体

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US5580434A (en) * 1996-02-29 1996-12-03 Hewlett-Packard Company Interface apparatus for capillary electrophoresis to a matrix-assisted-laser-desorption-ionization mass spectrometer
US20030220447A1 (en) * 1996-09-26 2003-11-27 Shearwater Corporation Soluble, degradable poly (ethylene glycol) derivatives for controllable release of bound molecules into solution
US7452715B1 (en) * 1999-06-25 2008-11-18 Wyeth Holdings Corporation Production of the lipidated form of the peptidoglycan associated lipoproteins of gram-negative bacteria
US20090060952A1 (en) * 1999-06-25 2009-03-05 Wyeth Holdings Production of the lipidated form of the peptidoglycan-associated liproproteins of gram-negative bacteria
US20070184524A1 (en) * 2000-11-20 2007-08-09 Gokarn Ravi R 3-hydroxypropionic acid and other organic compounds
JP2005117251A (ja) * 2003-10-06 2005-04-28 Sanyo Electric Co Ltd 通信装置
US20070069122A1 (en) * 2005-06-03 2007-03-29 Angelique Augustin In situ polypeptide identification

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